Current therapies for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are unsatisfactory, During the past year, we and three other groups discovered that U2AF1 and seven other components of the pre-mRNA splicing complex are recurrently mutated in patients with MDS and AML. Small molecule modulators of this complex have,been developed by our collaborators and others. The most advanced compounds in this class (called sudemycins) are soluble, stable, active against tumor xenografts in mice, non-toxic to normal cells, and can be synthesized in gram quantities. Sudemycins bind the SF3b splicing complex and induce apoptosis in cell lines after brief exposure, in part by causing alternative splicing of MDM2, a negative regulator of P53. The long-term goal of this proiect is to develop RNA splicing modulators as targeted therapv for MDS and AML, Our preliminary data demonstrate enhanced sensitivity of cells expressing mutant U2AF1 to sudemycin. We hypothesize that mutations in RNA splicing machinery confer sensitivity to splicing modulators by dysreguiating expression of alternatively spliced products that contribute to MDS/AML pathogenesis. We will test this hypothesis using well-defined systems in vitro and in vivo. In Specific Aim 1, primary human AML myeloblasts with or without splicing gene mutations, based on completed sequencing of all coding genes in these samples, will be used to assess the impact of sudemycins (and other splicing modulators) on growth, viability, apoptosis, and cell cycle kinetics in vitro and chimerism, apoptosis, and trilineage differentiation in vivo after xenotransplantation into immune deficient mice. This will determine whether splicing gene mutations serve as biomarkers of sensitivity to these drugs. In Specific Aim 2, we will discover alterations in the transcriptome induced by splicing modulators and develop sensitive and quantitative assays for assessment of the on-target effects of these agents. This project addresses SPORE translational endpoints by identifying genetic biomarkers that are predictive of response to splicing modulators and by developing assays that will be used for pharmacodynamic monitoring in clinical trials of splicing modulators: RELEVANCE (See instructions): Current chemotherapy treatments cure a minority of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We and others have recently discovered that RNA splicing, a critical metabolic pathway, is altered in many of these patients and compounds that modulate this pathway are available. This project will test the activity of this novel class of compounds in human cells and develop assays and preclinical data needed to initiate clinical trials of these agents in patients with MDS or AML.